Cell Biology of Vasopressin-Induced Water Channels

Massachusetts General Hospital, Boston MA

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Abstract

Description (provided by applicant) The major objectives of this renewal are to identify the intracellular pathways and mechanisms of regulated apical versus basolateral AQP2 insertion and recycling, to characterize cytoskeletal and other accessory protein interactions that are involved in AQP2 trafficking, and to define novel CAMP-independent signaling events leading to AQP2 membrane insertion that might be exploited in vivo to bypass vasopressin receptor (V2R) signaling. All of our aims are based on original published and unpublished work that was achieved during the previous funding period of this Program Project. Our specific aims are: 1) To determine the contribution of direct versus indirect AQP2 membrane delivery and the intracellular pathways followed by recycling AQP2 using immunocytochemistry, biotinylation and membrane fractionation in cultured cells, kidney slices, whole animals and real-time fluorescence microscopy. The role of Arf GTPases, dynamin and Hcs70 in AQP2 recycling will be determined using transfected cell models. We recently found that Hsc70 interacts with the AQP2 C-terminus. We will look for differential expression of these and other key targeting proteins in principal cells that insert AQP2 into different membrane domains in situ, by laser capture microdissection and DNA array screening; 2) To characterize cytoskeletal (actin, tubulin) and other accessory protein interactions that are involved in AQP2 trafficking, and to determine the effect of AQP2 phosphorylation on these interactions. Real-time vesicle translocation assays will use isolated microtubules and AQP2-GFP-expressing vesicles. The effect of Rho-family GTPases on membrane insertion and recycling of, phospho- and non-phospho AQP2 will be determined in transfected cells. The role of the newly-discovered interaction between the AQP2-C-tail and a Cdc42-GAP (GTPase activating protein) will be examined using biochemical assays of GAP activity. We will continue our search for AQP2 interacting proteins using pull-down assays with GST-AQP2 fusion proteins and mass spectrometry, phage display and two hybrid screening; 3) To characterize and determine the physiological significance of novel cAMP-independent signaling pathways leading to AQP2 membrane insertion. We showed that cGMP elevation (by atrial natriuretic factor and NO) results in AQP2 membrane insertion, and we will determine the pathway by which this occurs using specific PKA and PKG inhibitors (e.g., does PKG phosphorylate AQP2 directly in situ?). We will determine the ability of cGMP phosphodiesterase inhibitors (e. g., SildenafiUViagra) to stimulate AQP2 membrane insertion in vitro and in vivo, as a potential strategy of bypassing the V2R defect that results in X-linked nephrogenic diabetes insipidus. These aims will be achieved using a coordinated approach that involves the use of purified, recombinant AQP2 domains (e.g., the C-terminus), transfected cell lines expressing AQP2 and GFP-AQP2, slices of kidney tissue in vitro, and finally whole animal studies to examine the physiological significance of the AQP2 targeting and signaling pathways that we uncover. We once again anticipate that significant interaction with our colleagues within the Program will occur, and that our work will benefit greatly from the related studies proposed in their individual proposals.